Method of collecting protein substances having biological activity with respect to the nervous system comprising extraction of protein from submaxillary glands

ABSTRACT

A PROCESS FOR THE RECOVERY OF PROTEIN SUBSTANCES HAVING A BIOLOGICAL ACTIVITY TO PROMOTE THE GROWTH AND DEVELOPMENT OF THE NERVOUS SYSTEM WHICH COMPRISES (A) EXTRACTING THE SUBMAXILLARY GLANDS OF LARGE ANIMALS, I.E. BOVINE, EQUINE AND SWINE, WITH AN AQUEOUS EXTRACTING AGENT SELECTED FROM WATER AND SALINE SOLUTION; (B) ADDING TO THE EXTRACTED SOLUTION SULFOSALICYLIC ACID OR SODIUM SALT TO FORM A SOLUTION HAVING A CONCENTRATION OF 1-2% AND ADJUSTING THE PH OF THE SOLUTION TO 3.0-4.0 AND COLLECTING THE RESULTING PRECIPITATES; (C) DISSOLVING SUCH COLLECTED PRECIPITATES IN AN AQUEOUS SOLUTION; AND (D) SUBJECTING THE AQUEOUS SOLUTION FORMED IN (C) TO FRACTIONAL PRECIPITATION WITH ACETONE OR ALCOHOL IN A CONCENTRATION OF 40-50% AND COLLECTING THE FRACTIONAL PRECIPITATES CONTAINING THE PROTEIN SUBSTANCE. A PROTEIN SUBSTANCE (S) AND A PROTEIN SUBSTANCE (F) CAN BE FURTHER PRODUCED BY ACRINOL PRECIPITATION OF THE REDISSOLVED PROTEIN SUBSTANCE.

Die. 26, 1972 MASAAKI YAMAMOTO ET AL 3,707,531

METHOD OF COLLECTING PROTEIN SUBSTANCES HAVING BIOLOGICAL ACTIVITY WITH RESPECT TO THE NERVOUS SYSTEM COMPRISING I EXTRACTION OF PROTEIN FROM SUBMAXILLARY GLANDS Original Filed March 1; 1966 2 SheetS Sheet 1 Fig. IC

Fig.lB

Fig. IA

Bat. 26, 1912 MA5AAK| YAMAMOTO ET AL 3,701,531

METHOD OF COLLECTING PROTEIN SUBSTANCES HAVING BIOLOGICAL ACTIVITY WITH RESPECT To THE NERVOUS SYSTEM COMPRISING EXTRACTION OF PROTEIN FROM SUBMAXILLARY GLANDS Original Filed March 1. 1966 2 Sheets-Sheet 2 LL] FRACTION F 5 0 FR CTION S m d g 2 OONTROII- l l LL! 0 Ct LL! ED 2 D Z 24 48 72 T20 hrs United States Patent 3,707,531 METHOD OF COLLECTING PROTEIN SUB- STANCES HAVING BIOLOGICAL ACTIVITY WITH RESPECT TO THE NERVOUS SYSTEM COMPRISING EXTRACTION OF PROTEIN FROM SUBMAXILLARY GLANDS Masaaki Yamamoto, Kawasaki, and Tsuneyuki Nakazawa, Omiya, Japan, assignors to Teikoku Hormone Mfg. Co., Ltd., Tokyo, Japan Division of application Ser. No. 838,025, June 19, 1969, now Patent No. 3,549,610, which is a continuation of application Ser. No. 531,001, Mar. 1, 1966, now zgagnfioned. This application Aug. 4, 1970, Ser. No.

5 Claims priority, application Japan, Mar. 3, 1965, 40/ 12,278 Int. Cl. C07g 7/00 US. Cl. 260-412 R 3 Claims ABSTRACT OF THE DISCLOSURE A process for the recovery of protein substances having a biological activity to promote the growth and development of the nervous system which comprises (a) extracting the submaxillary glands of large animals, i.e. bovine, equine and swine, with an aqueous extracting agent selected from water and saline solution; (b) adding to the extracted solution sulfosalicylic acid or sodium salt to form a solution having a concentration of 1-2% and adjusting the pH of the solution to 3.0-4.0 and collecting the resulting precipitates; (c) dissolving such collected precipitates in an aqueous solution; and (d) subjecting the aqueous solution formed in (c) to fractional precipitation with acetone or alcohol in a concentration of 40-50% and collecting the fractional precipitates containing the protein substance. A protein substance (S) and a protein substance (P) can be further produced by acrinol precipitation of the redissolved protein substance.

This application is a division of application Ser. No. 838,025 filed June 19, 1969, now US. Pat. 3,549,610, which application in turn is a continuation of application Ser. No. 531,001 filed Mar. 1, 1966, now abandoned.

This invention relates to a method of collecting from the submaxillary glands of large animals protein substances which possess biological activity with respect to the nervous system (hereinafter referred to as active substances).

In the past, numerous biologically active substances, for example, substances which promote the growth and development of the nervous system have been found in the various organs of rodents such as, say, mouse and in snake venom.

For instance, in Levi-Montalcini: Regional Neurochemistry, Pergamon, New York, p. 362 (1960) and S. Cohen: Proc. Natl. Acad. Sci., US, 46, 302 (1960), it is disclosed that biologically active substances are extracted from either mouse submaxillary glands or snake venom, and that the active substances are proteins which possess moreover unique biological activities.

There is however a statement at p. 377 of the aforementioned Regional Neurochemistry that this nervegrowth promoting factor was not detected at all in the submaxilliary glands of the large animals, such as bovine.

And in accordance with the hitherto commonly held view in the field of biology, thepresence of such active substances in the various organs of large animals such as particularly bovine, equine and swine, was held to be highly improbable.

However, when, in accordance with the conventional methods, mouse submaxillary glands and snake venom are used as the raw materials, much difficulty is encountered in employing the aforesaid active substances clinically, since the source of the foregoing raw materials are extremely restricted.

It is therefore a primary object of the present invention to provide a method of collecting from readily available and low-cost raw materials protein substances which possess biological activity with respect to the nervous system.

Another object of this invention is to provide a method whereby protein substances having biologically active effects can be obtained from the submaxillary glands of large animals, particularly bovine, equine or swine, by removing the large amount of mucoid present therein and hence making it possible to obtain the protein substances with high purity and moreover in relatively large amounts on a commercial scale.

A further object of this invention is to provide protein substances having pronounced therapeutic effects on such disorders of the nervous system as peripheral neuropathy, neuralgia, neuritis, introxications neuropathy, cerebrovascular diseases, demyelating disease, myelitis, cerbical spondylosis and migraine.

An additional object of the invention is to provide a method of administering the aforesaid protein substances to the human body.

Other objects and advantages of this invention will be apparent from the following description.

The foregoing objects are attained in accordance with the present invention by a method of collecting protein substances having biological activity with respect to the nervous system, which comprises extracting with an aqueous extracting agent the submaxillary glands of large animals, inclusive of bovine, equine and swine, adding to this extraction solution a protein precipitant which can reversibly redissolve the precipitate to deposit as a precipitate a fraction consisting predominantly of said protein substances having biological activity with respect to the nervous system, and thereafter isolating from said solution said substances which have been substantially purified.

The important feature of the present invention resides in the unexpected discovery of the fact that protein substances which could promote the growth and development of the nervous system were present in relatively large amounts in the submaxillary glands of large animals such as bovine, equine and swine, which heretofore was considered as not containing these active substances.

On the other hand, a considerably large amount of mucoid is also present in the submaxillary glands of these large animals, for example, an amount so large as to exceed times that of the active substances. Owing to the presence of this large amount of mucoid, the isolation of the active substances cannot possibly be accomplished even though the conventional method of extracting the nerve growth promoting factor from the mouse submaxillary glands or snake venom is employed.

In spite of this fact, we also unexpectedly found that when the extracting procedure to be hereinafter fully described is employed, the complete separation of the protein substances having biological activity with respect to the nervous system, from the aforesaid large amount of mucoid could be achieved.

PREPARATION OF THE AQUEOUS EXTRACTION SOLUTION FROM SUBMAXILLARY GLANDS According to the present invention, the submaxillary glands of large animals, particularly bovine, equine or swine, are used as the raw material. In general, it is preferred that the submaxillary gland material to be used is in a fresh condition. Much difiiculty is however involved in collecting fresh submaxillary glands when they are to be collected in large amounts on a commercial scale. In the preferred mode of operating this invention, purified active protein substances, as hereinafter described, can be prepared even from submaxillary glands which have been stored for a long period of time. Hence, according to this invention, the freshness of the submaxillary glands to be used is not of great concern.

Submaxillary glands, which have been collected from the carcasses of large animals, after having been removed of their fatty and other tissues, are rubbed and ground and thereafter extracted by using as the aqueous extracting agent either water or a dilute aqueous solution of an inorganic salt such, for example, as a saline solution of a concentration of 2% by weight. The insoluble matter is then removed. Any of the known extracting procedures customary in the art can be used. In this case, it is preferred that the extracting agent be used in an amount which is from three to five-fold that of the submaxillary glands on a weight basis and that the pH of the aqueous extracting agent is 7.0 to 8.0. The so obtained extraction solution contains a small amount of the active protein substances and a large amount of mucoid, for example, from 100 to 500-fold that of the active protein substances. Hence, in accordance with this invention, the protein substances consisting predominantly of the active substances and the large amount of mucoid are separated from the extraction solution by the following fractional operation.

SEPAR'AFPION FROM THE EXTRACTION SO-LU- TION OF THE PROTEINS CONSISI ING PRE- DOMI'NANTLY OF THE ACTIVE SUBSTANCES In order to separate the large amount of mucoid and the proteins consisting predominantly of the active substances, which are contained in the foregoing extraction solution, one of the following operations is generally employed, the fraction of the aforesaid protein substances being collected from the extraction solution as a precipitate.

(A) The proteins consisting predominantly of the active substances are selectively deposited as a precipitate, while the mucoid remained in the solution.

(B) The mucoid is first deposited as a precipitate and removed following which the proteins consisting predominantly of the active substances are collected from the remaining extraction solution as a fractional precipitate.

Therefore, the term, protein precipitant, as used herein and in the appended claims, is meant to refer to a precipitant which is capable of precipitating the proteins without denaturing them permanently and moreover has the actions described in (A) and (B), above. As such a precipitant, included are a wide range of water-soluble inorganic salts, certain of the organic acids and the water-soluble organic solvents. Suitable protein precipitants are the sulfates represented by such as ammonium sulfate, sodium sulfate and magnesium sulfate, the water-soluble zinc salts such as zinc chloride and zinc acetate, the organic acids represented by such as sulfosalicylic acid, and the water-soluble organic solvents such as ac to e and asshole For purpose of illustration, typical protein precipitants having the aforesaid actions of (A) and (B) will be described below.

(A) Zinc chloride is added to the extraction solution in an amount such that the concentration of the zinc chloride in the solution does not exceed 0.02 mol, and preferably a range between 0.0067 and 0.014 mol. In this case, the active protein substances form a complex with the zinc salt. Now, if, as required, a water-soluble organic solvent such as acetone or alcohol is added to this system, the zinc complex is readily precipitated. On the other hand, since the large amount of mucoid remains in the solution without binding with the zinc salts, the separation of the two components can be readily accomplished. The conjoint use in this case of such water-soluble solvents as acetone or alcohol is particularly desirable for accelerating the precipitation of the zinc complex and for reducing the amount of the zinc salt to be used and facilitating the redissolution of the zinc complex.

According to this mode, there is the advantage that the separation and removal of the mucoid can be effectively carried out even when submaxillary glands which have been stored over a long period of time are to be used as the raw material. The reason is that in the case of such stored submaxillary glands the mucoid is denatured and hence in a state that precipitation thereof does not readily take place. Consequently, it becomes difficult to eliminate the mucoid from the extraction solution in advance. However, when the procedure here described is employed, the proteins consisting predominantly of the active substances are deposited as a zinc complex precipitate and thus the admixture of mucoid in the product is very small.

(B) First, when ammonium sulfate is added to the extraction solution and the concentration of the salt in the solution and its pH are maintained between a certain range, a major portion of the mucoid in the solution is precipitated, and the proteins consisting predominantly of active substances remain in the solution. In the case of ammonium sulfate, the salt concentration of the solution is, in general, preferably below 24% by weight (below 0.5 saturation). The pH range suitable for precipitat ing the mucoid in the solution will vary depending upon the class of the submaxillary glands used and operating conditions. Generally, in the case of that of bovine or equine, the mucoid precipitates between pH 7.0-7.5 and in the case of swine, between pH 4.0-4.5. Alternatively, in the case of an extraction solution of the submaxillary glands of swine, the mucoid can be removed by an operation consisting of adding the ammonium sulfate, adjusting the pH to 6.5-7.0, then adding an organic solvent such as acetone or the like and eliminating the clots of the mucoid, and thereafter decanting the bottommost layer of the three layers of the solution formed when it is allowed to stand.

When to the extraction solution from which the mucoid has been so removed ammonium sulfate is again added and the salt concentration of the solution is increased, proteins consisting predominantly of the active substances are precipitated. It is preferred that the concentration of the ammonium sulfate in the solution at this time be about 33% by weight (0.7 saturation). In this manner, the fraction of proteins consisting predominantly of the active substances can be isolated as a fractional precipitate in a concentration of the ammonium sulfate from 0.5 to 0.7 saturation in the solution.

Numerous other inorganic salts besides those mentioned above can be used as the protein precipitant. While the concentration of the salt used will vary depending upon the kinds of the salt, the range of concentration for precipitating only the fraction of the proteins consisting predominantly of the active substances can be determined readily on the basis of a simple experiment. It is possible to use as the protein precipitant the water-soluble organic 91Y11t$ such as alcohol and acetone and organic acids,

such as sulfosalicylic acid, in the form of their sodium salts. For example, the fraction of the proteins consisting predominantly of the active substances can be collected as a precipitate by adding either alcohol or acetone such that the concentration thereof in the solution becomes 30-50% by volume. Alternatively, by adding sulfosalicylic acid to the extraction solution such that the concentration thereof in the solution becomes 1-2%, the fraction of the active substances can be similarly collected as a precipitate between pH 3-4. The organic solvents are however preferably used as a precipitant along with the aforesaid inorganic salts, or as a precipitant when the protein fraction consisting predominantly of the active substances, after having been precipitated by the addition of the inorganic salt or organic acid, are again subjected to fractional precipitation.

According to this invention, the so obtained precipitate is again dissolved in an aqueous medium. This aqueous medium may be either water, an aqueous solution of an inorganic salt of pH 3 to 10, or an aqueous solution containing a dissociating agent of the protein-inorganic salt complex. When an organic solvent, organic acid or inorganic salt is used as the protein precipitant, the resulting precipitate can readily be dissolved in water or the foregoing aqueous solution of an inorganic salt. On the other hand, when a protein complex forming agent, such as a water-soluble zinc salt, is used as the protein precipitant, a dissociating agent, such as ethylene diamine tetraacetic acid, an alkali metal salt of citric acid or glycine, is used for redissolving the resulting precipitate.

Needless to say, the purity of the active protein substances can be enhanced by repeating the hereinabovedescribed operation for at least two times. Further, this is most convenient for the preparation of protein substances having biological activity with respect to the nervous system of high potency. For example, in the case Where the aforesaid precipitate still contains a considerable amount of mucoid or impure proteins, a water-soluble organic solvent such as acetone is added as a prec p tant to the redissolved solution, and a fractional precipitate of an acetone concentration in the solution of below 50% by volume is collected. When an inorganic salt such as ammonium sulfate has been used as the precipitant in this case, it is preferred that the ammonium sulfate be eliminated by means of such as dialysis prior to adding the acetone to the redissolved solution.

On the other hand, when submaxillary glands which have been stored for a long period of time are to be used, protein substances consisting predominantly of the active substances of still greater purity can be obtained by the following procedure. First, a zinc salt such as zinc chloride is added as a precipitant to the extraction solution, then the resulting zinc complex is redissolved, followed by the addition of ammonium sulfate to the solution of the redissolved zinccomplex to deposit a precipitate, and thereafter dialyzing this precipitate, after which acetone is added as a precipitant to the dialyzate solution, thereby obtaining the proteins consisting predominantly of the active substances.

ISOLATION OF THE ACTIVE SUBSTANCES FROM THE PROTEINS CONSISTING PREDOMINANTLY OF THE ACTIVE SUBSTANCES From the solution containing proteins consisting predominantly of the active substances from which mucoid has been substantially eliminated, as hereinabove described, the substantially purified active substances are isolated by any of the optional means. According to this 1nvention, a particularly preferred procedure consists of adding a water-soluble organic solvent, such as alcohol or acetone, to the aforesaid solution and collecting the fractional precipitate with acetone in the concentration between 40% and 50% by volume in acetone concentration of the total volume of the solution. This is because the protein substances having biological activity precipitate at the acetone concentration between 40% and 50% by volume, whereas the other proteins either precipitate at below 40% by volume or remain in the solution.

Thus, substantially purified protein substances having biological activity with respect to the nervous system can be obtained according to this invention. These substances can be used, as such for such disorders of the nervous system as peripheral neuropathy, neuralgia, neuritis, intoxications neuropathy, cerebrovascular diseases, demyelating disease, myelitis, nervous disturbances in accompanying with Osteopathy, cervical spondylosis, migraine and other disturbances and disorders of the nervous system.

According to our studies, it was found however that the electrophoretic pattern of these active substances showed that they were made up principally of two components having dififerent mobilities. For selectively separating these two components, the fractional precipitation by means of acrinol is effective. Namely, the aforesaid protein substances obtained by the fractional precipitation with acetone are dissolved in a saline solution, and a weakly basic aqueous acrinol solution is added gradually in increment to the foregoing solution while separating the forming precipitate, the addition of the acrinol solution being such that the weight ratio of the protein to acrinol becomes finally 4:1. Thus, by fractionating this solution into a mother liquor containing a protein substance (fraction S) which is especially effective With respect to the growth of neurons and the regeneration of axons and a precipitate consisting of an acrinol complex of a protein substance (fraction F) effective with respect to the proliferation of the glial cells, it is possible to separate the active substances into the foregoing protein substance (S) and protein substance (F).

The active substances, which have been collected from the submaxillary glands of the large animals such as bovine, equine or swine, have been purified to a high degree in the hereinbefore-described purification process, but for attaining still greater homogeneity, they are caused to be adsorbed to the two ion exchange agents of CM-cellulose or DEAE-cellulose in a low concentration of salt and are eluted within a specific pH range by raising the salt concentration. Thus can be obtained active substances which have been homogenized to a high degree.

PROPERTIES OF THE ACTIVE PROTEIN SUBSTANCE Both active substances (S) and (F), which have been so separated, have biological activity with respect to the nervous system, and it was found that the substance (S) was particularly effective for promoting the growth of the nerve cells and regeneration of the axons, while substance (F) exhibited pronounced effects in promoting the proliferation of the glial cells.

The purified active substances, both (S) and (F), obtained by the invention method exhibit the usual protein color reaction, their maximum absorption being at 279 m and 278 mg, respectively. They are nondialytic, are decomposed by proteolytic enzymes, and are unstable to heat. It is thus clear that they possess the properties of protein. They are judged to be practically a single component from analysis by electrophoresis and gas chromatography and their purity is high. The yield of the active substance (S) from the glands varies somewhat depending upon the animal from which the submaxillary glands are obtained, but is roughly a value of -200 mg. per kg. of the glands. In the case of bovine, it is -150 mg. per kg. of submaxillary glands; in the case of swine, 100-120 mg. and in the case of equine, -200 mg. On the other hand, the yield of the active substance (P) is 500 mg. per kg. of submaxillary glands, a comparable yield being obtained in the case of swine and equine.

By way of illustration, the physical and chemical pro ganglia, spinal cord and brain stem from chicken embryo erties of the homogeneous active substances (S) and (F) obtained from bovine are shown in Table I, below.

and the promotive efiect of the growth of the nerve tissues was examined, using the axon length as the index,

TAB LE I (Physical and chemical properties of bovine S-iraction and F-iraction] S-fraction F-fraction Homogeneity:

Electrophoresis Homogeneous (pH 4.0 and pH 8.6) Homogeneous.

Ultracentrifugation. (pH 8 6) Do. Sedimentation coefficient- Difiusion coetficient Molecular weight Ultraviolet absorption. Isoelectric point Sugar content Phosphorus eonten 0.1 Protein color reaction Negligible. Lipoprotein color reaction Negligible" Do. Susceptibility for protease Hydrolyzed, loss activity.. Loss activity. Antigenic action The active protein (S) which is obtained by the invention method difiers from the Nerve Growth Promoting Factor which Levi extracted from the mouse submaxillary glands in the point that the molecular weight it can be seen that, as shown in Table II, below, with a 48-72 hour culture the growth of the axons of the spinal ganglia is promoted 2-3.5 times as compared with the control.

TABLE II.EFFECT ON THE NERVOUS SYSTEM OF PURIFIED POWDER EXTRACTS OF VARIOUS CLASSES OF SUBMAXILLARY GLANDS Extracted mouse submaxillary glands by the conventional method. The culture fluid alone without the addition of the active substance.

of the latter is 44,000 whereas that of the former is 84,000, a figure about twice as large.

The preparations from the submaxillary glands of bovine or equine of a blood serum phosphorus reducing substance and a parotinlike substance having the property of reducing the blood serum calcium have been known hitherto (for example, Japanese patent application publication Nos. 4,747/1957 and 4,598/1957). However, the isoelectric points of the foregoing substances are 4.5 and 5.4, respectively, whereas the fraction-F of the active substances of this invention, as is apparent from the foregoing table, has an isoelectric point of 6.4, while the fraction-S has that of 5.0. Hence, it is clear that the two differ as protein substances. In addition, their biological activities also diir'er entirely. Furthermore, in the above methods either the fractional precipitates at the alcohol concentrations from 60% to 80% by volume or the isoelectric point precipitate at pH 4.5 or 5.4 is being collected, whereas in the present invention when alcohol or acetone is used as the precipitant, the collection of the precipitate is made at a concentration of these precipitants of below 50% by volume. And not only the active substances are not deposited as an isoelectric point precipitate as noted hereinabove, but also when inorganic salts are used as the precipitant, their use is made with their pH in the neighborhood of neutrality. Therefore there is a definite diiference in the extraction methods themselves between the foregoing published methods and those of this invention.

THE BIOLOGICAL EFFECTS ON THE NERVOUS SYSTEM OF THE ACTIVE SUBSTANCES The powders of the active substances so obtained possess the effects of promoting to a marked degree the growth of the nerve cells, regeneration of the axons and proliferation of the glia cells.

(A) The effect of active substance (S) When active substance (S) was added to a culture flu d ll a issue cultu e m thod with su h t s ues as s inal Thus, it is seen from Table II, above, that (1) -Active substance S obtained from any source according to this invention has the eflect of promoting the growth of the peripheral sensory ganglia rather than the central spinal cord and brain stem; and

(2) According to the culture method, the growth promoting effect of the active substance obtained by the method of this invention far surpasses that of the mouse NGF obtained by the prior Cohen method. Further, in the case of the metabolic promoters including vitamins, their effect on axon growth is inferior and the cannot attain in a short period of time an effect which is lasting for a long period as in the case with the active substance S of this invention.

Again, when the nerve cells are cultivated for a long period of time and the eifect of preventing the degeneration and disintegration of the neurons is examined, it can be seen, as shown in Table III, that active substance S had the effect of preventing the increase in the degeneration of the nerve cells at the end of one as well as two weeks of cultivation. Thus, it is seen that it has the eifect of maintaining the nerve cells in a sound state.

TABLE III.EFFECT ON THE NERVE CELLS OF PURIFIED ACTIVE SUBSTANCE IN THE CASE OF LONG TERM CULTURE Purified active substance admin- Control (unadministered (bovine,

It is evident from the foregoing clincial examples that excellent eifects such as the following are obtained when the active protein substances of this invention have been administered; namely, 1) that excellent therapeutic effects are seen in the case of disorders of the nervous system, particularly disorders of the sensory nerves; (2) that the period required for the cure can be shortened greatly as compared with the hitherto-employed vitamins B and B12, nicontinic acid preparations, adrenocortical hormone and salicylic acid preparations; (3) that they may be used independently; (4) that there is observed a tendency to alleviation of the condition of illness with injections administered two or three times even in those cases where symptoms do not disappear, thus having remarkable effects in the initial stages of the illness; (5) that they are capable of being administered to patients of all ages; and (6) that the treatment period till disappearance of symptoms can also be shortened in the case of intoxications neuropathy.

Thus, when summarized, the present invention possesses such remarkable features in that it has found the presence in the submaxillary glands of the large animals, such as bovine, equine and swine, of protein substances which are biologically active with respect to the nervous system and has made possible the separation of said protein substances from the mucoid present in large quantities in said submaxillary glands, thereby providing a method of preparing said active protein substances in great quantities on a commercial scale without the need for any special operations or equipment.

Furthermore, the active protein substances obtained from the submaxillary glands of bovine, equine or swine in accordance with this invention have such advantages as that they possess great afiinity for the nervous system, particularly the peripheral nerves, that they have effects which promote to a marked degree the growth of neurons, reinervation of the axon and proliferation of the glial cells, and that when they are administered intramuscularly to the human body, early therapeutic effects are observed in the case especially of disorders of the peripheral nerves.

This invention will be further illustrated by the following examples which are intended in an illustrative sense and not in limitation of the invention.

EXAMPLE 1 Submaxillary glands of bovine, swine or equine are removed of fatty and other tissues and then rubbed and ground. Extraction of the glands is carried out for 3 hours by adding a threefold amount of 2% saline to 1 kg. of each and maintaining pH 7.0-8.0 with NaOH. After separating the extraction solutions, an equal amount of 2% saline solution is added to the extraction residue and the extraction is repeated for one hour under identical conditions, following which the extraction solutions are separated and combined with the extraction solutions obtained first. To each of these extraction solutions is added a small amount of solid ammonium sulfate to obtain 0.5 saturation (24%), the pH then being adjusted to 7.0-7.5 with ammonia water in the case of extraction solution of bovine or equine, and to pH 4.0-4.5 with HCI in the case of the extraction solution of swine, after which the solutions are left standing in a refrigerator overnight. The deposited precipitates are centrifuged. To the clear mother liquors are further added solid ammonium sulfate to obtain 0.7 saturation (33% and the pH is adjusted to 7.0-7.5 with ammonia water in the case of the bovine or equine solution and to 4.0-4.5 with HCl in the case of the swine solution, followed by standing in a refrigerator overnight. On the following day, a thin bed of Celite is made on a Buchner funnel and the deposited precipitates are separated by filtering the solution with suction. The precipitates remaining on the Buchner funnel are extracted twice along with the Celite with about 200 ml. of water. Then 14 after combining the extraction solutions and submitting to dialysis against running water for 2-3 days, the red dialisand liquids within a dialyzing bag are centrifuged. After elimination of the small quantity of insoluble matter, they are then submitted to acetone fractionation.

To about 500 ml. of the clear liquids within a dialyzing bag, an equal amount of acetone is added with stirring under cooling condition to obtain a 50% concentration, the depositing precipitates being then collected by centrifuging. The precipitates, as obtained, are extracted and dissolved by adding thereto a 50-fold portion of 2% saline solution. Acetone is added to the nearly clear solutions with stirring under cooling condition to make 40% concentration. To the supernatant liquids obtained after removing the deposited precipitates, acetone is added in a similar manner to make 50% concentration. The precipitates which are formed between this 40 and 50% concentratives are collected by centrifuging, after which they are dehydrated with acetone and ether, and then dried to obtain the powder of active protein substances. The yield was about the same for bovine, swine or equine, being respectively 1-1.5 grams.

0.9% saline solution is added to these protein powders and the powders are dissolved to prepare solutions whose protein concentration is 2%. Acrinol aqueous solution prepared in advance is then added dropwise to the protein solutions with stirring under cooling condition until the concentration of 0.143% (ratio to the protein of 1:10) is obtained, followed by standing the solutions still for one hour in a refrigerator after adjusting the pH of the solutions to 9.5 with NaOH. The precipitates [active substance (F)] separating out are removed by centrifuging. The 0.5% acrinol solution is added in a similar manner to the supernatant liquids until the concentration of 0.186% (1:7) is obtained, after which the pH is adjusted to 9.5. Similar operations are repeated successively with acrinol concentrations of 0.222% (1:5) and 0.248% (1:4), the pH in both instances being adjusted to 9.5, and the depositing precipitates [active substance (F)] and separated to finally obtain 0.248% supernatant liquids.

A small quantity of active carbon or Amberlite XE- 64, which are washed in advance, are added to the foregoing liquid to adsorb the acrinol. Then when suction filtration is carried out in a Buchner funnel having a Celite bed provided thereon, there are obtained pinkish tinged clear filtrates from which acrinol is completely eliminated. HCl is added dropwise to the filtrates to adjust the pH to 6.5-7.5, after which acetone is added with stirring under cooling condition, and the precipitates separating between 40 and 50% are collected by centrifuging. After dissolving the precipitates in a small portion of water and dialyzing against distilled water, they are freezed and dried, whereby highly purified active substance (S) is obtained. The yield was about the same for bovine, swine and equine, being respectively 120-150 mg., mg. and -200 mg.

The active substance (F) is obtained by collecting the aforesaid precipitates which were deposited by the addition of acrinol, extracting with a threefold portion of 1 M-ammonium acetate bulfer solution (pH 9.5), adding a threefold portion of ethanol to the centrifugal upper layer liquids and depositing precipitates, dissolving these precipitates in 0.9% saline solution, followed by adding a threefold of acetone to the solutions and thereafter drying the separating precipitates. The yield of this purified active substance (F) was the same for bovine, swine and equine, being 500 mg. per kilogram of the glands.

EXAMPLE 2 be achieved by repeating the hereinafter described column chromatography.

First, either CM-cellulose or CM-sephadex is thoroughly washed in advance and then packed in a suitable column. An aqueous solution of the S-component of F- component is passed through this column and thereafter washed with water. The ineffective and colored component is eliminated by adsorption, while the respective active substances are contained in the unadsorbed effluent. Next, either DEAE-cellulose or DEAE-sephadex is thoroughly washed in advance, and a column buffered with a phosphoric acid buffer solution of pH 6.0 is prepared. The foregoing effluent of the S-component or F-component is passed through this column, followed by washing with 0.01 M phosphoric acid buffer solution of a pH 6.0. The respective active substances remain adsorbed to the column. The column is eluted succesively with 0.01 M phosphoric acid buffer solution of pH 6.0 and efiiuents to which have been added NaCl in the amounts of 0.12 M and 0.5 M-0.75 M. The S-component is eluted from the column with the efiiuent of 0.12 M NaCl concentration, while the F-component is eluted from the column with the effluent of 0.5-0.75 M NaCl concentration. A twofold portion of acetone is added to the respective effluents to deposit the respective precipitates, which are dissolved in a small portion of water and dialyzed to remove the salts which are admixed. This chromatography by means of DEAE-cellulose or DEAE-sephadex is repeated once more, and finally the S- and F-co-mponents are freezed and dried and thus homogeneous products of both are obtained.

The yield of the homogeneous S-component is 50-80 mg. per kg. of glands of all the foregoing animals, and that of the F-component is 200-300 mg.

EXAMPLE 3 Swine submaxillary glands are treated as in Example 1, after which the extraction of one kg. thereof is repeated twice, and then after combining the extraction solutions, solid ammonium sulfate is added until 0.5 saturation (24%) is obtained. After adjusting the pH of the solution to 6.5-7.0 by adding NaOH, acetone is added with vigorous stirring while being cooled until the concentration becomes 30%, whereupon mucoid clo-ts begin to separate during the interim, and at the end point, the clots become completely separated from the solution and float. These mucoid clots are gathered and removed from the solution. The solution is then allowed to stand still in a refrigerator overnight, whereupon the solution separates into three layers. The active substances are contained in the bottommost layer which exhibits a red color. This bottom layer liquid is taken out by decantation, to which is then added solid ammonium sulfate until 0.7 saturation (33%) is obtained, after which it is adjusted to pH 4.0-4.5 and then allowed to stand overnight in a refrigerator. The following day, it is suction filtered as in Example 1 by preparing a thin bed of Celite above a Buchner funnel, after which the precipitate is extracted with Water and dialyzed against running water, whereby is obtained a dialyzate solution containing the active substances of red color.

Thereafter, acetone fractionation is carried out as in Example 1 to obtain 1.5 grams of the powder of active substances. This powder is then purified by the acrinol fractionation method and the active substances (F) and (S) are separated. The yield was 100-120 mg., which was comparable to that of the swine gland in Example 1.

EXAMPLE 4 To extraction solutions obtained from the submaxillary glands of bovine, swine or equine, acetone is added by the procedure described in Example 1 until it becomes 50% by volume in the solutions, after which the solutions are allowed to stand overnight in a refrigerator. The depositing precipitates are taken out by means of centrifuging. The precipitates are then dissolved and extracted by adding a fourfold ortion of 0.9% NaCl olu ion. Ther f e 16 by carrying out the ammonium sulfate fractionation, dialysis and acetone fractionation treatments as in Example 1, similar results were obtained.

EXAMPLE 5 Solid sodium sulfate is added to the extraction solutions obtained from the submaxillary glands of bovine, swine or equine by the procedure described in Example 1 until 0.5 saturation (15%) is obtained at 30 C., after which the pH is adjusted to 7.0-7.5 with ammonia water in the case of those bovine or equine and to 4.0 with HCl in the case of swine. The solutions are then allowed to stand overnight at 30 C. The mucoid precipitate separating out is removed by centrifuging, and to the clear mother liquors, is solid sodium sulfate further added at 30 C. until 0.7 saturation (22%) is obtained. The pH is adjusted to 7.07.5 in the case of those of bovine or equine and to 4.0 in the case of that of swine, following which the solutions are allowed to stand overnight at 30 C. The depositing precipitates are separated by suction filtration after which the precipitates are dissolved in a small portion of water and dialyzed against running water for 2-3 days. The hydrolyzate liquid within a dialyzing bag is then treated as in Example 1. Thus, purified active substances are obtained as in Example 1 and at near comparable yields.

EXAMPLE 6 To extraction solutions obtained from the submaxillary glands of bovine, swine or equine as in Example 1, are added 20% sodium sulfosalicylate aqueous solution which had been prepared by neutralizing with NaOH, the additions being in amounts '1/10-1/20 until a concentration of roughly 1-2% is obtained. Next, the pH is adjusted to 3.0-4.0 by adding dropwise 20% HCl, then the solutions are allowed to stand in a refrigerator for 30-60 minutes, and thereafter centrifuging is conducted after adding a small amount of Celite. The upper layer liquid is discarded and a fourfold portion of Water is added to the precipitates and extraction is carried out after adjusting the pH to 7-8 with NaOH. This is followed by filtration, whereby clear filtrates are obtained. The filtrates are treated with ion exchange resin to eliminate the sodium sulfosalicylate and thereafter acetone fractionation is carried out as in Example 1. Thus are obtained the active substances with yields and purities comparable to those obtained in Example 1.

EXAMPLE 7 Nonfresh submaxillary glands for bovine, equine and swine which had been stored for the various periods of time indicated in Table V, below, are removed of their fatty and other tissues and rubbed and ground. To 1 kg. of each is added a threefold portion of 2% saline solution, and the extraction thereof is carried out for 3 hours while maintaining the pH between 7.0-8.0 with NaOH. After separating the extraction solutions, 2% saline solution is added to the extraction residues in an equal amount and the extraction is repeated for 1 hour under identical conditions, after which the extraction solutions are separated and combined with their respective first extraction solutions. Separately, zinc chloride is dissolved in water to make a concentration of 0.1-0.2 mol, to 1 part of which solution are added 4 parts of acetone to prepare an aqueous acetone mixture containing zinc chloride. The zinc concentration of this mixed solution is 0002-0004 mol and its acetone concentration is This mixed acetone solution is added to the foregoing mixed extraction solutions in a ratio of 1 to 2, and without adjusting the pH the solutions are allowed to stand in a refrigerator overnight. The zinc concentration of these solutions is 00067-0014 mol and the acetone concentration is 26.7%. On the following day, the deposited precipitates are separated by either centrifuging or by preparing a thin Celite bed on a Buehner funnel and uction filtering.

These precipitates are dissolved by adding thereto a threefold portion of either 1.5% sodium citrate, 1% glycine or 1% EDTA solution, followed by filtering to eliminate the Celite and thereafter centrifuging, whereupon clear solutions containing the active substances are obtained. From these solutions, fractional precipitate is collected, as described in Example 1, under conditions with ammonium sulfate 0.5-0.7 saturation in neutral solution in the case of bovine or equine and similar frac tional precipitate in a weakly acid in the case of swine. Then after dialysis, first precipitates are obtained by making acetone concentration 50% from the dialyzate solutions. When these precipitates in their state as-obtained are extracted with 2% saline solution and the precipitates which are separated out between 40-50% acetone concentration are dried, powders of crude active substances containing very little mucoid, whose purification process is easy, can be obtained. The results obtained are shown in Table V, below, to compare with the results obtained in Example 1.

() dissolving the collected precipitates of (b) in an aqueous medium; and

(d) subjecting the solution of (c) to the fractional precipitation with acetone or alcohol in a concentration between 40% and 50% and collecting the fractional precipitates containing a protein substance.

2. The process of claim 1 wherein a protein substance (S) effective in promoting the growth of neurons and axons is recovered by the steps including dissolving the collected precipitates of step (d) in an aqueous medium, adding acrinol incrementally to the resulting solution until the acrinol concentration is 0.248% by weight; recovering the resultant precipitate from the solution; and recovering said protein substance (S) from said solution.

3. The process of claim 1 wherein a protein substance (F) eifective in promoting of myelines is recovered by the steps including dissolving the collected precipitates of step (d) in an aqueous medium; adding acrinol incrementally until the acrinol concentration is 0.248% by weight;

TABLE V.MUCOID CONTENTS OF CRUDE POWDERS EXTRACIED BY THE ZINC- ACETONE METHOD Example 7 Example 1 Content of Content of reducing Yield index reducing Yield index Storage Crude powder sugar in of active Crude powder sugar in of active period yield (per kg. crude powder substances in yield (per kg. crude powder substances in (25 C.) of gland), g. (percent) crude powder of gland), g. (percent) crude powder 0 1-1. 1-2 103 1-1. 5 3-5 100 2 weeks 1-1. 5 1-2 103 1-1. 5 3-5 100 1 month. ll. 5 1-2 103 1-1. 5 5-7 98 2 months 1-1. 5 1-2 103 3 months ll. 2 2-3 82 0. 8-1. 2 10-12 74 4 months 0.8-1. 2 2-3 82 0. 8-1. 2 72 6 months 0. 8-1. 2 2-3 82 The yield index of the active substances is represented by a value calculated on a basis of a yield of 100 for that of zero day storage in Example 1.

We claim:

1. A process for the recovery of protein substances having a biological activity to promote the growth and development of the nervous system by the fractional precipitation from the submaxillary glands of large animals selected from the group consisting of bovine, equine and swine, which comprises:

(a) extracting said glands with an aqueous extracting agent selected from the group consisting of water and saline solution;

(b) adding to the extracted solution of (a) sulfosalicylic acid or a sodium salt of sulfosalicylic acid to form a solution having a concentration of 12%, ad-

OTHER REFERENCES Annals of New York Academy of Sciences, vol. 85, 1960, Ito, pp. 228-229, 233-235, 265-272, 274, and 306.

General Biochemistry, Fruton, 1960, p. 108.

justing the pH of the solution to 3.0-4.0 and collect- HOWARD E. SCHAIN, Primary Examiner ing the resulting precipitates therefrom; 

